A number of spermatogenesis-specific gene products have been described in mammals, including autosomal phosphoglycerate kinase (PGK-2). In general the pattern of expression of such genes has been analyzed at the protein level, with very little work on the transcriptional level. Furthermore essentially no data has been reported concerning mechanisms of regulation and/or induction of such genes. There are several unique questions with respect to spermatogenesis-specific genes concerning the relationship of their expression with: (1) onset of meiosis (including the question of haploid expression); (2) inactivation of the single X-chromosome; and/or (3) any other testis-specific factors including specific hormonal environments. The PGK gene system of the mouse is ideal to analyze these questions. PGK-2 is an autosomal spermatogenesis-specific gene. Its expression and regulation can be compared to that of PGK-1 which is X-linked and ubiquitously expressed in all somatic cells. In order to do this cDNA and genomic clones of both PGK-2 and PGK-1 will be isolated and their nucleotide sequences determined. Based on this information synthetic PGK-1- and PGK-2-specific probes will be produced and used in conjunction with the technique of in situ cytohybridization to determine the exact pattern (cell type and stage) of PGK-2 versus PGK-1 transcription. In addition, in order to assess the role of 5-methylcytosine in control of PGK gene expression, the degree of methylation in CCGG sites in PGK-1 and PGK-2 coding and non-coding regions in different cell types at appropriate stages will be determined such that active and inactive PGK genes can be compared. The long range goal of the proposed research is to develop and understanding of the regulation and expression of spermatogenesis-specific genes, and a better understanding of the molecular mechanisms of genetic regulation in general.